CN102787105B - Cotton uridine diphosphate-glucose dehydrogenase gene UGD6, its encoding protein and application - Google Patents

Abstract

The invention relates to a cotton uridine diphosphate-glucose dehydrogenase (UGD) gene UGD6, its encoding protein and application. The cotton UGD has an amino acid sequence as shown in SEQ ID No.2 or an SEQ ID No.2 derived amino acid sequence that has an equivalent function to the above sequence and is formed by substitution, deletion or adding of one or several amino acids. The cotton UGD gene has an amino acid sequence as shown in SEQ ID No.1. By studying the expression patterns of the gene at different fiber development stages and transforming Arabidopsis thaliana, the gene is proved to play an important role in forming cell wall cellulose and hemicellulose precursors, and the materials directly participate in cotton fiber formation. Thus, with the gene, a new candidate gene for cotton fiber quality improvement is provided, and the gene is of great application value.

Description

Cotton UDP-glucose dehydrogenase gene UGD6, its proteins encoded and application

Technical field

The present invention relates to the genetically engineered field, particularly relate to cotton UDP-glucose dehydrogenase gene, its proteins encoded and application.

Background technology

Cotton fibre is one of natural resource that known fiber element purity is the highest.Cotton fibre quality mainly comprises the proterties such as staple length, intensity, mic value, and these proterties are controlled by genotype own mainly.China's cotton fibre exists interior quality relatively poor, and fibre strength is than problems such as U.S. cotton are low.Therefore, carry out the research of molecular biology and the control of related gene expression of cotton fiber development, the genetically engineered improving cotton fiber quality is significant for utilizing.

Cotton fiber is in fact kind of a fur, is formed by upper epidermic cell Development And Differentiation by the ovule that is positioned at ovary is outer.From on form, the growth course of cotton fiber cell is the process of the extraordinary elongation of cell and the extraordinary thickening of cell walls.The formation of cotton fiber cell generally can be divided into four periods that the phase mutual is overlapping: fiber initiating cell differentiation projection, elongating stage, secondary wall thickening phase, dehydration ripening stage.Elongating stage and secondary wall thickening phase are the critical periods that cotton fibre quality forms.Primary cell wall synthetic is one of key activities of cell elongation phase, directly related with staple length.Before finishing elongating stage, fibrocyte starts the synthetic of secondary cell wall, is mainly cellulosic deposit, and the thickening of fibrocyte secondary wall determines fiber strength.

Cotton fibre cell walls mainly comprises the non-cellulose compositions such as Mierocrystalline cellulose and xyloglucan, xylan, pectin polysaccharide.Content of cellulose accounts for 95% of secondary wall dry weight; The non-cellulose material forms the main component of primary wall.Uridine diphosphoglucose (UDP-Glc) is the synthetic substrate of Mierocrystalline cellulose, and UDP-Glc is synthetic cellulose under the cellulose synthase effect.Non-cellulosic polysaccharide is polymerized by different monose, only has after they are activated formation nucleosides sugar, could be as the synthetic substrate of non-cellulosic polysaccharide.The synthetic of nucleosides sugar completed by a whole set of enzyme, research finds that nearly all nucleosides sugar all take UDP-Glc and GDP-Man(guanosine diphosphte mannose) through the associated metabolic enzyme catalysis, synthesize for precursor, therefore, nucleosides sugar conversion involved enzyme plays an important role the synthetic of cotton fibre cell walls Mierocrystalline cellulose and non-cellulose.

Uridine diphosphate glucuronate (UDP-GlcA) is the key precursor thing during much nucleosides sugar synthesizes, by inference, about 50% cell wall substance derives from precursor UDP-GlcA, it is the product that UDP-glucose dehydrogenase (UDP-glucose dehydrogenase, UGD) catalysis UDP-Glc forms.Therefore, UDP-glucose dehydrogenase is the key enzyme that forms non-cellulosic polysaccharide and pectin.The clone of existing many pieces of relevant UGD genes report on plant.At first the UGD gene clones out in soybean, subsequently, in Arabidopis thaliana, sugarcane, corn, clone respectively this gene.Research shows, UGD is in different species and tissue, and its expression level has larger difference.UGD mainly expresses in spire and immature xylem in willow, in ripe phloem almost without expressing.Yet in Arabidopsis thaliana Seedlings, mainly at root, express, and mainly in vascular system, express in ripe plant.Ramie UGD mainly expresses in stem, secondly, in phloem and leaf, in root, express minimum.Soybean UGD has a large amount to express in main root point and lateral root, in main root, climax leaves, only have trace expression.Illustrate that UGD has very high expression at the tender tissue of children, and expression amount is seldom in plant mature tissue, this illustrates that this enzyme plays very important effect in the process that forms cell walls Mierocrystalline cellulose and hemicellulose precursor, the expression of UGD will exert an influence to cell walls Mierocrystalline cellulose and non-cellulosic polysaccharide content, and and then have influence on cellulose microfibril and non-cellulose is crosslinked.In sum, the UGD gene is significant for the inherent mechanism that discloses cotton fibre quality formation, has a good application prospect in the fibrous quality genetic improvement.UGD plays an important role in the cotton fibre building-up process, but so far in cotton still without the further investigation.

Summary of the invention

The object of the invention is to provide a kind of cotton UDP-glucose dehydrogenase gene (UGD6) and proteins encoded thereof, be used to improving cotton fibre quality.

Another purpose of the present invention is to provide the carrier that contains said gene.

Another object of the present invention is to provide the host cell that contains said gene or carrier.

Still a further object of the present invention is to provide cotton UDP-glucose dehydrogenase or the application of cotton UGD6 gene in improving cotton fibre quality.

The albumen of cotton UGD6 genes encoding of the present invention, have the aminoacid sequence shown in SEQ ID NO.2 or this sequence through replacing one or several amino acids formed aminoacid sequence with same function.

The nucleotide sequence of cotton UGD6 gene of the present invention as shown in SEQ ID No.1, total length 1912bp, the long 1443bp of ORF, the long 168bp of 5 ' UTR, the long 301bp of 3 ' UTR.

Particularly, the present invention is based on four UDP-glucose dehydrogenase (At1g26570 of Arabidopis thaliana, AT3g29360, At5g15490 and At5g39320) encoding sequence, utilize BLASTN in the expressed sequence tag database of the state-run biotechnology information center of U.S. website (http://www.ncbi.nlm.nih.gov/) cotton, to search for the higher est sequence of similarity, in conjunction with electronic splicing, RT-PCR and RACE technology clone, obtain cotton UDP-glucose dehydrogenase gene, called after UGD6.

Should be appreciated that the degeneracy of considering codon, for example can be in its coding region, under the condition that does not change aminoacid sequence, or at its non-coding region under the condition that does not affect protein expression, the gene order of the above-mentioned albumen of encoding is modified.Therefore, the present invention also comprises replacement, the interpolation that the gene order of the above-mentioned albumen of encoding is carried out and/or lacks one or more Nucleotide, has the nucleotide sequence that has identical function with above-mentioned encoding gene.The present invention also comprises just sequence or the antisense sequences based on described gene, comprise cloning vector or the expression vector that contains described nucleotide sequence or its fragment, the host cell that contains described carrier, utilize described host cell to prepare method of cotton UDP-glucose dehydrogenase etc.

Described this sequence is through replacing one or several amino acids formed aminoacid sequence with same function, refers to that site outside the active function territory of this sequence carries out the sequence that limited amino acid whose protection replaces gained and still can keep original activity.The active function territory of aminoacid sequence of the present invention is 209 ~ 306, and replaceable one or several amino acid obtains having the aminoacid sequence of same function outside this site.Such as, SEQ ID NO.3 is depicted as the nucleotide sequence of sea island cotton UGD7 gene, SEQ ID NO.4 is depicted as the aminoacid sequence of UGD7 genes encoding, with the UGD6 aminoacid sequence, compare, 26th, 40,56,60,64,149,193,202,368 9 amino acid whose replacements have occurred, and have same functional effect.

Utilize Real-time PCR to detect the expression amount of UGD6 gene in upland cotton " No. 8, CCRI " and sea island cotton " Pima90-53 " cotton fiber development different times and seedling root, stem and leaf, the variation tendency of finding this gene expression amount in two kinds is similar, after blooming, continue high efficient expression in the fiber of 20 ~ 30d, on a declining curve after 30d, illustrate that it has a certain effect to the initial sum of cotton fibre secondary wall is synthetic.In the 15d seedling, the UGD6 gene, in the tissue of seedling root, hypocotyl and the leaf of " No. 8, CCRI " and " Pima90-53 ", all has similar expression amount, and expression amount difference is: stem>root>leaf.

The UGD6 gene is connected with prokaryotic expression carrier pET-32a, transforms the bacillus coli DH 5 alpha competent cell, obtain the colibacillus engineering strain that contains recombinant plasmid.Under the IPTG inductive condition, expressed fusion protein, and carry out the SDS-PAGE electrophoretic analysis.The pET32a-UGD6 recombinant plasmid specific band occurs on the position of about 68.4kDa, successfully expressing protein in prokaryotic cell prokaryocyte of this gene is described.

The UGD6 gene is connected with fusion expression vector pCamE ∷ GFP, proceeds to the bacillus coli DH 5 alpha competent cell, obtain the colibacillus engineering strain that contains recombinant plasmid.By particle gun, bombard onion epidermis cell.After dark the cultivation, in the lower observation of microscope (blue-light excited), determine that the protein localization of this genes encoding is in cytoplasmic membrane system.

Adopt the Agrobacterium flower-dipping method by S-UGD6(justice) and the A-UGD6(antisense) expression vector proceeds in Columbia type Arabidopis thaliana, turns that UGD enzymic activity in S-UGD6 and A-UGD6 Arabidopis thaliana is corresponding to be increased and reduce.Turn A-UGD6 Arabidopis thaliana plant content of cellulose and compare significantly and increase with wild-type, turn the S-UGD6 Arabidopis thaliana and significantly descend.Turn A-UGD6 gene Arabidopis thaliana stem length and stem and slightly all significantly be less than wild-type, straw stiffness significantly diminishes, and turns S-UGD Arabidopis thaliana phenotype without considerable change.

Extract wild-type and transgenic arabidopsis RNA, carry out the Real-time pcr analysis, find the importing of UGD gene and knock out, remarkably influenced the expression level of the endogenous UGD gene of Arabidopis thaliana, and UGD is as the key enzyme of UDPG metabolism, can be to nucleosides sugar synthetic and even the synthetic of Mierocrystalline cellulose and pectin exert an influence.

Beneficial effect of the present invention:

(1) cotton UGD6 gene provided by the invention provides new candidate gene for cotton fibre quality improvement genetically engineered.

(2) cotton UGD6 gene provided by the invention is in the different cotton seed of fiber strength, the expression amount variation tendency is variant, the UGD6 gene is at cotton fiber development up-regulated expression elongating stage, its peak expression after blooming 20 ~ 30 days, can be used for non-cellulosic polysaccharide and the secondary wall Mierocrystalline cellulose study on the synthesis of primary wall.

(3) cotton UGD6 gene provided by the invention and cotton fiber development have important relationship, thereby can utilize the further converting cotton of genetic engineering means to improve cotton fibre quality.

The accompanying drawing explanation

Fig. 1 is opening code-reading frame (open reading frame, ORF) the RT-PCR amplification of cotton UGD6 gene of the present invention, and wherein, M is DL5000Marker, and 1 is UGD6 ORF amplified production.

Fig. 2 is the present invention finds cotton UGD6 gene conserved domain on NCBI result.

Fig. 3 is cotton UGD6 gene 5 ' RACE of the present invention and 3 ' RACE amplified production, and wherein, M is DL2000Marker, and 1 is 5 ' RACE amplified production, and 2 is 3 ' RACE amplified production.

Fig. 4 is the expression analysis result of cotton UGD6 gene of the present invention at the Fibre Development different times; X-coordinate DPA is the different number of days after blooming, and ordinate zou is the expression amount of UGD6 gene.

Fig. 5 is the expression analysis result of cotton UGD6 gene of the present invention in root, stem and leaf.

Fig. 6 is the E.coli BL21(DE3 that turns cotton UGD6 gene) induce before and induce the SDS-PAGE detected result of rear purpose expressing fusion protein, arrow is depicted as the purpose fusion rotein, wherein, M: protein standard molecular weight, the total protein that the empty bacterial strain of 1:BL21 (DE3) plys is induced without IPTG, the total protein that 2:pET-32a (+) empty carrier is induced without IPTG; The total protein that 3:UGD6-pET-32a (+) induces without IPTG; The total protein of the empty bacterial strain of 4:BL21 (DE3) plys after IPTG induces 6 hours; The total protein of 5:pET-32a (+) empty carrier after IPTG induces 6 hours; The total protein of 6:UGD6-pET-32a (+) after IPTG induces 6 hours.

Fig. 7 is the result of UGD6 gene Subcellular Localization of the present invention, wherein, and the cell under A:pCamE-GFP is blue-light excited; Cell under the visible light of B:A; Cell under C, D, F, G:pCamEUGD6 ∷ GFP are blue-light excited; Cell under E:C and D visible light.Cell under the visible light of H:F and G.

Fig. 8 is that cotton UGD6 gene eukaryotic expression vector of the present invention builds schematic diagram, wherein, and A: plasmid pBI121 structure; The B:pBI121-SUGD6 structure; The C:pBI121-AUGD6 structure.

Fig. 9 is the seedling of cotton UGD6 gene transformation Arabidopis thaliana of the present invention, wherein, and A: turn the S-UGD6 Arabidopis thaliana; B: turn the A-UGD6 Arabidopis thaliana.

Figure 10 turns A-UGD6 T for growth 10 days ₃In generation, is relatively individual with wild-type Arabidopis thaliana plant, wherein, and WT: the contrast of wild-type Arabidopis thaliana, AU: turn A-UGD6 Arabidopis thaliana plant.

Figure 11 turns A-UGD6 gene T for growth 10 days ₃In generation, is relatively long with the root of wild-type Arabidopis thaliana, wherein, and WT: the contrast of wild-type Arabidopis thaliana, AU: turn A-UGD6 Arabidopis thaliana plant.

Figure 12 turns A-UGD6 gene T for growth 35 days ₃In generation, compare with the phenotype of wild-type Arabidopis thaliana, wherein, and WT: the contrast of wild-type Arabidopis thaliana, AU: turn A-UGD6 Arabidopis thaliana plant.

Figure 13 is the comparison diagram that turns S-UGD6 and S-UGD6 Arabidopis thaliana strain and wild-type content of cellulose, wherein, and WT: the contrast of wild-type Arabidopis thaliana, SU-: turn S-UGD6 Arabidopis thaliana T ₃For different strains, AU-: turn A-UGD6 Arabidopis thaliana T ₃For different strains.

Figure 14 is the comparison diagram that turns S-UGD6 and A-UGD6 Arabidopis thaliana strain and wild-type UGD enzymic activity, wherein, and WT: the contrast of wild-type Arabidopis thaliana, SU-: turn S-UGD6 Arabidopis thaliana T ₃For different strains, AU-: turn A-UGD6 Arabidopis thaliana T ₃For different strains.

Embodiment

Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.

Carrier of the present invention, bacterial strain, plasmid all is commercially available.

The clone of embodiment 1 cotton UGD6 gene

(1) at first utilize four uridine diphosphate glucuronate decarboxylase (At1g26570 of Arabidopis thaliana, AT3g29360, At5g15490 and At5g39320) encoding sequence, utilize tBLASTN in NCBI cotton expressed sequence tag database, to search for the higher est sequence of similarity, utilize DNASTAR software to carry out electronic splicing to the est sequence that retrieval obtains, obtain the splicing sequence UGD6 with complete open reading frame.

(2) according to splicing sequences Design Auele Specific Primer, at the upstream and downstream primer, introduce respectively KpnI and SalI restriction enzyme site (underscore part) simultaneously.The upstream and downstream primer of UGD6 gene is respectively:

Upstream primer: 5 '- GGTACCATGGTGAAGATCTGTTGC-3 '

Downstream primer: 5 '- GTCGACTTATGCCACTGCAGGC-3 '

(3) utilize the Auele Specific Primer of design to take respectively cotton fibre cDNA that upland cotton " No. 8, CCRI " and sea island cotton " Pima90-53 " (purchased from the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute's National Cotton germ plasm resource storehouse in mid-term) bloomed latter 20 days and carry out pcr amplification, acquisition UGD6 gene purpose band (Fig. 1) as template;

The PCR product of the UGD6 gene (4) recovery obtained is connected with pGM-T, adopt the pGM-T clone test kit clone purpose fragment of TIANGEN Biotech (Beijing) Co., Ltd., connecting product adopts the heat shock conversion method to proceed to intestinal bacteria TOP10 competent cell (purchased from sky root biochemical technology company limited), and screening positive clone, through Shanghai biotechnology company limited, check order, the row that check order consistent with expected results, and gene order indifference between two cotton seeds.UGD6 gene ORF sequence is shown in the long 1443bp of SEQ ID NO.1(), corresponding aminoacid sequence is SEQ ID NO.2.

Similarity analysis and the conserved structure domain analysis of embodiment 2 cotton UGD6 gene coded proteins

Utilize DNAMAN to carry out similarity analysis to the aminoacid sequence of cotton UGD6 gene, cotton GhUGD1 ~ UGD5 and 4 Arabidopis thaliana UDPG dehydrogenase gene proteins encoded, result shows that the amino acid sequence similarity of cotton UGD6 gene and cotton UDPG dehydrogenase gene proteins encoded is distributed between 89.0% ~ 99.6%, and the amino acid sequence similarity of Arabidopis thaliana UDPG dehydrogenase gene proteins encoded is distributed between 84.4% ~ 92.7%, illustrate that cotton UGD6 gene belongs to UDPG acidohydrogenase gene family.

Utilize NCBI(http: //www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) the CDD database is guarded territory BLAST analysis online, found UDPG/GDP-mannitol dehydrogenase gene family conserved regions (Fig. 2), illustrated that UGD6 gene of the present invention belongs to UDPG dehydrogenase gene family.

The clone of embodiment 3 cotton UGD6 gene 5 's and 3 ' non-translational region (UTR) sequence

Take cotton UGD6 gene ORF sequence as basis, by the requirement of Clontech RACE test kit, design respectively 5 ' RACE and 3 ' RACE primer, concrete primer sequence is as follows:

5 ' RACE primer:

GSP：5'-CAGCTCCGAGCCCCCGAGTCTTGGT-3'

NGSP：5'-TCACTGTTCCAGGCAGCGATCCTAG-3'

3 ' RACE primer:

GSP：5'-ATATATGACCCGCAGGTGACCGAAG-3'

NGSP：5'-GCAACCCATGAGTCCCACGACTGTC-3'

The cotton fibre cDNA that upland cotton " No. 8, CCRI " and sea island cotton " Pima90-53 " bloomed latter 20 days of take is material, by Clontech RACE test kit operation steps, complete 5 ' end and 3 ' end non-translational region sequence amplification, obtain the purpose band, the long 420bp of UGD6 5 ' RACE, the long 530bp(Fig. 3 of 3 ' RACE).The PCR product is checked order, and ultimate analysis obtains the long 168bp of UGD6 5 ' UTR, the long 301bp of 3 ' UTR.By sequencing result and the splicing of UGD6 gene open reading frame, obtain full length cDNA sequence, and gene order indifference (SEQ ID NO.1) between two cotton seeds.

The spatial and temporal expression pattern analysis of embodiment 4 cotton UGD6 genes

Use the RNAplant of TIANGEN Biotech (Beijing) Co., Ltd. to extract test kit and extract " No. 8, CCRI " and sea island cotton " Pima90-53 " ovule and the fiber RNA of 5,10,15,20,25,30,35,40 days (days post anthesis, DPA) afterwards of blooming on the same day of blooming.The plant total RNA extraction reagent box of the rich biotech company of Beijing Aurion is used in the extraction of Radix Gossypii, stem and leaf RNA.

The precious biotech firm in employing Dalian

RT reagent Kit With gDNAEraser (Perfect Real Time) test kit synthesizes the strand cDNA in above 9 periods, concentration Beckman DU800 spectrophotometric determination.Cotton UGD6 gene is carried out to the Real-time pcr analysis in the expression of cotton fiber development different times.Internal reference is selected EF1 α, and its primer sequence is as follows:

EF1αF：5′-GCTGAGATGAACAAGAGGTCATTC-3′

EF1αR：5′-GGAATCAATAATCAAAACAGCACAG-3′

Real-timePCR result (Fig. 4) demonstration, cotton UGD6 gene continue high efficient expression in the fiber of 20 ~ 30DPA, on a declining curve after 30d, illustrate that UGD6 synthesizes and has certain effect the initial sum of cotton fibre secondary wall; In the 15d seedling, the UGD6 gene all has expression in the Radix Gossypii cauline leaf, and expression amount is the highest in stem, takes second place in root, minimum in leaf (Fig. 5).

Structure and the abduction delivering of embodiment 5UGD6 prokaryotic expression vector

(1) acquisition of the structure of prokaryotic expression carrier and transformant

Utilize KpnI and SalI respectively ORF and prokaryotic expression carrier pET-32a (+) plasmid (purchased from Novagen company) of cloning the UGD6 gene obtained in embodiment 1 to be carried out double digestion and reclaim the purpose fragment.

Expression vector is connected with the goal gene fragment, transform bacillus coli DH 5 alpha, through PCR and enzyme, cut detection, screening contains the positive colony of recombinant plasmid, utilize the plasmid that the heat shock method will contain positive colony to proceed to BL21(DE3) in bacterial strain (purchased from sky root biochemical technology company limited), and further picking positive colony, through plasmid extraction, enzyme, cut to detect to obtain and contain recombinant plasmid pET-32a(+)-the colibacillus engineering strain of UGD6.

(2) abduction delivering of recombinant protein and evaluation

To contain above all types of recombinant plasmid and contain the BL21(DE3 of empty plasmid (contrast)) bacterial strain is at 28 ℃, the IPTG final concentration is after under the condition of 1.0mmol/L, inducing 6h, carry out the rectilinear gel electrophoresis of SDS-PAGE, wherein separation gel is 12%, and concentrated glue is 5%.

Result as shown in Figure 6, is removed cross-film district recombinant plasmid pET-32a(+ through IPTG containing of inducing)-e. coli bl21 (DE3) of UGD6 contrasts and compares with containing empty plasmid, the difference band that expression amount obviously increases occurs at 68.4KDa.The effable UGD6 purpose of Bioinformatics Prediction fusion rotein comprises UGD6 albumen (about 48KDa) and pET-32a label protein (about 20.4KDa), and molecular weight is approximately 68.4KDa.Therefore, this difference band is pET-32a(+)-the UGD6 gene is expressed in e. coli bl21 (DE3) purpose fusion rotein.

The Subcellular Localization of embodiment 6UGD6 fusion expression vector structure and fusion rotein

(1) structure of fusion expression vector

Be designed for the primer of vector construction hexose transport protein.Restriction enzyme site SalI and Kpn I (underscore part) are added in ORF two ends at the UGD6 gene, and primer sequence is as follows:

PUF：5’- GTCGACATGGTGAAGATCTGTTGC-3’

PUR：5’- GGTACCTGCCACTGCAGGCAT-3’

With this primer, carry out the PCR reaction, and reclaim the purpose fragment, be connected with the pGM-T carrier, the thermal shock method transforms competent escherichia coli cell DH5 α, and picking positive colony, extraction plasmid carry out enzyme and cut detection, order-checking, obtain intermediate carrier pGM-UGD6.

Intermediate carrier pGM-UGD6 and expression vector pCamE ∷ GFP are used respectively to SalI and Kpn I double digestion, the electrophoresis detection enzyme is cut product, reclaiming the purpose fragment connects, the thermal shock method transforms competent escherichia coli cell DH5 α, the picking positive colony, carry out plasmid extraction, enzyme is cut and is detected and order-checking, obtains fusion expression vector pCamUGD6 ∷ GFP.

(2) Subcellular Localization of particle gun mediation

The sterilizing filter paper of MS culture medium flat plate upper berth one deck good, be laid in dull and stereotyped central authorities by the onion entocuticle, bombards and transform fusion expression vector by particle gun.Culturing room observes and takes a picture after secretly cultivating 1-3d under microscope (blue-light excited).

Onion epidermis cell after micro-Microscopic observation particle gun bombardment, find that the carrier pCamE-GFP that does not contain foreign gene is positioned at (Fig. 7 A-7B) on nucleus and cytolemma, consistent with forefathers' positioning result.PCamE-UGD6 ∷ GFP green fluorescence appears on the structures such as cytolemma, nuclear membrane, vacuole skin and endoplasmic reticulum, has observed in addition the motion of obvious film bubble, and UGD6 protein localization (Fig. 7 C-H) on cell membrane system is described.

Embodiment 7 cotton UGD6 gene eukaryotic expression vectors build and the Arabidopis thaliana genetic transformation

(1) structure of carrier for expression of eukaryon

Fig. 8 is shown in by the construction of eukaryotic expression vector schematic diagram.Be designed for the primer that builds plant expression vector, add restriction enzyme site SacI and Xba I (underscore part) at the ORF two ends of UGD6 gene, sequence is as follows:

SUF：5’- TCTAGAATGGTGAAGATCTGTTGC-3’

SUR：5’- GAGCTCTTATGCCACTGCAGG-3’

AUF：5’- GAGCTCATGGTGAAGATCTGTTGC-3’

AUR：5’- TCTAGATTATGCCACTGCAGGCAT-3’

With this primer, carry out the PCR reaction, and recovery purpose fragment, with the pGM-T carrier, be connected, the thermal shock method transforms competent escherichia coli cell DH5 α (day root biochemical technology company limited product), the picking positive colony extracts plasmid, through enzyme, cut and detect and check order, obtain intermediate carrier pGM-SUGD6 and pGM-AUGD6.

Intermediate carrier and carrier for expression of eukaryon pBI121p35S are used respectively to SacI and Xba I double digestion, electrophoresis enzyme is cut product, reclaim the purpose fragment, then connect, the thermal shock method transforms competent escherichia coli cell DH5 α, and the picking positive colony, carry out plasmid extraction, through enzyme, cut and detect and order-checking, obtain carrier for expression of eukaryon pBI121p35S::SUGD6 and pBI121p35S::AUGD6.

(2) functional analysis of Arabidopis thaliana genetic transformation and UGD6

1) Arabidopis thaliana genetic transformation: plasmid pBI121p35S::SUGD6 and pBI121p35S::AUGD6 are transformed to agrobacterium strains GV3101 competent cell, adopt the Agrobacterium flower-dipping method that S-UGD6 and A-UGD6 carrier are transformed to Columbia type Arabidopis thaliana.Arabidopsis thaliana transformation plant inflorescence is soaked in to the conversion medium that contains goal gene, after several minutes, take out, moisturizing is watered sufficient nutritive medium after secretly cultivating 24h, the recovery normal illumination is cultivated, and treats the Arabidopis thaliana seed maturity, collects seed and carries out drying also after vernalization, plant on the MS screening culture medium (kantlex concentration is 100mg/L), screening obtains transgenic positive seedling (Fig. 9), transplants in vermiculite and cultivates, and the performing PCR of going forward side by side detects further determines the transgenic positive plant.Afterwards, then through 2 plantations of taking turns, screening until obtain T ₃For positive Arabidopis thaliana transgenic line, carry out phenotype and cell wall constituent analysis.

2) phenotype analytical: transgenic arabidopsis is compared with the wild-type Arabidopis thaliana, phenotype generation noticeable change, antisense UGD6 Arabidopis thaliana hypoevolutism, long (the Figure 10 that significantly shortens of root, Figure 11), the bolting time is than wild-type late (Figure 12), and the long and stem of stem slightly all significantly is shorter than wild-type, straw stiffness also significantly diminish (table 1).WT is the contrast of wild-type Arabidopis thaliana, and AU-is for turning A-UGD6 Arabidopis thaliana T ₃For different strains.And the adopted UGD6 Arabidopis thaliana phenotype of becoming a full member is without considerable change.

Table 1 turns the comparison of A-UGD6 Arabidopis thaliana strain and wild-type phenotype index

Strain	WT	AU-2	AU-3	AU-5
					Stem length/cm	26.78±1.07	14.98±0.80**	17.43±0.67**	18.25±1.29**

Stem is thick/mm	1.05±0.05	0.73±0.08**	0.77±0.07**	0.77±0.08**
					Straw stiffness/N	12.1±0.92	11.3±0.81**	9.9±0.68**	9.5±0.63**

3) cell wall constituent analysis: for further determining the function of UGD6 gene in cell wall polysaccharides transforms, extract the cell walls of transgenosis and wild-type Arabidopis thaliana, adopt cellulosic content in anthrone colorimetric method for determining arabidopsis cell wall.

Concrete steps are: Mierocrystalline cellulose in the mixing solutions boiling waterbath cell walls of employing acetic acid, nitric acid and water (8: 1: 2); ddH ₂O and washing with acetone final vacuum are drained; Sulfuric acid with 72% is dissolving cellulos again; Add anthrone reagent also fully after reaction, to measure A620, the contrast standard curve had both obtained the cell walls content of cellulose.

Result demonstration antisense UGD6 Arabidopis thaliana strain (AU) content of cellulose is compared significantly and is increased with wild-type, the significantly decline (Figure 13) of adopted UGD6 Arabidopis thaliana (SU) of becoming a full member.

4) UGD enzyme activity assay: extract the UGD enzyme of transgenosis and wild-type Arabidopis thaliana, get the Arabidopis thaliana in six weeks of growth by strain, extract proteolytic enzyme; Material is weighed, and adds protein extract (the 50mM Tris-HCl (pH7.5) of two volumes; 2mM EDTA, with before adding 5mMDTT), fully grind; 4 ℃, the centrifugal 2min of 11,200g, discard cell wall fragments, and supernatant recentrifuge 2min, be crude enzyme liquid.The purifying of UGD enzyme is used the gravity-type Sephadex desalting column (BSP090) of Shanghai biotechnology company limited to carry out the desalting and purifying of albumen.Purification step carries out in strict accordance with specification sheets.

Antisense UGD6 Arabidopis thaliana strain enzymic activity is compared remarkable reduction with wild-type, the adopted UGD6 Arabidopis thaliana plant of becoming a full member significantly increases (Figure 14).

The functional analysis result of UGD6 shows: the overexpression of UGD6 gene has significantly improved Arabidopis thaliana UGD enzymic activity, cause a large amount of uridine diphosphoglucose (UDP-Glc) to be consumed, and the Mierocrystalline cellulose of cell walls main ingredient is formed by glucan binding domian, the overexpression of UGD has caused the reduction of content of cellulose.

The inhibition of UGD gene is expressed, cause on the one hand a large amount of accumulations of UDP-Glc, cause increasing of content of cellulose, also directly caused on the other hand the minimizing by product uridine diphosphate glucuronate (UDP-GlcA) content of UGD catalysis glucose, most of sugar (arabinose, apiose, galacturonic acid and wood sugar etc.) directly or indirectly derives from UDP-GlcA in hemicellulose and pectin, broken the balance of original plant cell wall Mierocrystalline cellulose and non-cellulosic polysaccharide content, affect cell wall structure, thereby caused a series of phenotype to change.

5) expression analysis of the endogenous UGD gene of transgenic arabidopsis:

Extract the RNA of wild-type and transgenic arabidopsis, carry out Real-time PCR reaction, no matter found that foreign gene S-UGD6 and A-UGD6 all efficiently transcribe in Arabidopis thaliana, be to turn S-UGD6 or A-UGD6 gene, and the transcriptional level of the endogenous UGD gene of Arabidopis thaliana all significantly reduces.

The overexpression that result shows the UGD6 gene has produced the co-suppression effect to the expression of the endogenous UGD gene of Arabidopis thaliana, but the strong startup ability due to 35S promoter, external source UGD6 gene has obtained good expression, the acting in conjunction of endogenous and foreign gene, do not cause the considerable change of plant phenotype.The inhibition of UGD6 gene is expressed, and has broken the balance that in the Arabidopis thaliana, UGD expresses, and causes a series of phenotypic difference.

Therefore, excessive and the low scale of UGD6 gene reaches, cause content of cellulose in cotton fibre cell walls significantly to change, simultaneously may cause that the non-cellulosic polysaccharide material is the variation of xyloglucan, xylan or pectin polysaccharide content, the phenotypic characteristic that formation is similar to Arabidopis thaliana changes, and then have influence on cotton fiber length and intensity, affect cotton fibre quality.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (6)

1. the UDP-glucose dehydrogenase that grows cotton, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.2.

2. the gene of coding claim 1 described cotton UDP-glucose dehydrogenase.

3. gene according to claim 2, is characterized in that, its nucleotide sequence is as shown in SEQ ID No.1.

4. the carrier that contains the described gene of claim 2.

5. the host cell that contains the described gene of claim 2 or 3 or the described carrier of claim 4.

6. the described cotton UDP-glucose dehydrogenase of claim 1 or the described gene of claim 2 or 3 application in improving cotton fibre quality.

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